Department of Molecular Microbiology and Immunology, Oregon
Health Sciences University, Portland, Oregon
97201-3098,1 and Department of Biology,
California State University, Sacramento, California
95819-60772
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INTRODUCTION |
Salmonella enterica
serotype Typhimurium is a facultative intracellular pathogen that
causes a typhoid like disease in mice. Following oral infection,
bacteria actively invade the intestinal mucosa and enter the
bloodstream via the gut-associated lymphoid tissue (GALT). Subsequent
residence within professional phagocytes of the liver and spleen is
required for a persistent infection, which ultimately leads to the
death of the mouse. Growth and survival of Salmonella within
macrophages is supported by numerous studies, including the direct
observation of Salmonella within hepatic phagocytes
(45), comparative infection studies in genetic strains of
mice that produce macrophages with varying resistance to
Salmonella (38, 40), and the persistence of
infection in mice treated with gentamicin, an antibiotic that primarily
kills extracellular bacteria (10, 18). Finally, genetic
studies indicate that Salmonella mutants that are attenuated
for intramacrophage survival are also attenuated for systemic infection
in mice (20). While all of these studies demonstrate that
Salmonella survives and replicates within macrophages,
several groups have recently shown that Salmonella is also
able to kill these host cells (3, 13, 35, 39).
Contradictory results have been reported for Salmonella
genes required for the induction of apoptosis as well as the timing at
which it takes place. One study showed that serotype Typhimurium kills
macrophages as late as 18 h postinfection (35). This
process depends on the two-component regulatory system
ompR-envZ, as ompR was the only gene identified
in a stringent selection to find Salmonella mutants that are
unable to kill macrophages. InvA is an essential structural component
of the Salmonella pathogenicity island 1 (SPI1)-encoded type
III export apparatus, whereas SipB is a SPI1-secreted effector molecule
(22, 30). Null mutations in either invA or
sipB, two genes within SPI1, had no effect on the ability of
serotype Typhimurium to kill infected macrophages in this study
(35). However, other studies appear to contradict these
observations and demonstrate that within a few hours upon contact,
serotype Typhimurium induces apoptosis in infected macrophages in an
invA (and thus SPI1)-dependent process (13, 36,
39). SipB is both necessary and sufficient for the rapid
activation of this apoptotic pathway (29).
Here, we resolve this apparent controversy by demonstrating that
serotype Typhimurium kills macrophages via two independent processes.
It is demonstrated that SPI1 gene expression accounts for rapid
induction of apoptosis, whereas SPI1-independent, delayed induction of
apoptosis is abrogated in strains mutated in ompR and SPI2.
These results have important implications for understanding Salmonella pathogenesis, which are discussed.
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MATERIALS AND METHODS |
Bacterial strains, bacteriophages, and recombinant DNA
techniques.
Bacteria were grown overnight in Luria-Bertani (LB)
broth at 37°C. Antibiotics, when required, were used at the following concentrations: nalidixic acid (Nal), 50 µg/ml; chloramphenicol (Cam), 30 µg/ml; kanamycin (Kan), 60 µg/ml; and ampicillin (Amp), 100 µg/ml. Recombinant DNA techniques and Southern hybridizations were performed using standard protocols (4, 37).
Analytical-grade chemicals were purchased from Sigma (St. Louis, Mo.)
or Roche Biochemicals/Boehringer Mannheim (Indianapolis, Ind.).
Mutations in the ompR, invA, spiB, and
prc genes have been described previously (20, 23, 35,
49) and were used to construct a set of isogenic serotype
Typhimurium mutants (Table 1).
Bacteriophage KB1int was used to transduce the
ompR::MudJ allele of SWL350
(35) into SR-11 
3041 (wild type [wt]), yielding strain
AWM405 (ompR). Bacteriophage P22HTint was used to
transduce the invA::TnphoA allele of
AJB75 (7) into AWM501 (sipB, see below) and
AWM527 (ssrB, see below), yielding AWM544 (invA
sipB) and AWM545 (ssrB invA), respectively.
Bacteriophage P22HTint was used to transduce the
sipB::mTn5 allele of STN119
(49) into SR-11 3041 (wt), yielding strain AWM568
(sipB). Bacteriophage P22HTint was also used to
transduce the prc::Tn10 allele of
MS4290 (20) into SR-11 3041 (wt), yielding strain AWM664
(prc).
Allelic exchange was performed to disrupt the serotype Typhimurium
invA gene. An internal fragment of the invA gene
was amplified from serotype Typhimurium ATCC14028 (wt) using primers
5'-GCATGAATTCGCAGAACAGCGTCG-3' and
5'-GTTGTCTAGATCTTTTCCTTAATTAAGCC-3', which generated a PCR fragment with unique 5' EcoRI and 3' XbaI sites,
respectively. This PCR product was cloned into the EcoRV
site of pBluescript II SK(+) and sequenced. Subsequently, the
invA allele was inactivated by insertion of a
chloramphenicol resistance gene (a 1.2-kb SmaI fragment from
pCMXX [7]) into a unique internal SnaBI
site and cloned into suicide plasmid pKAS32 (48). The
resulting plasmid was electroporated into Escherichia coli
SM10
pir and conjugated to serotype Typhimurium ATCC 14028 derivative BA715 (rpsL) (1). A double crossover
at the invA allele was obtained via homologous recombination. A chloramphenicol- and streptomycin-resistant
exconjugant was selected and named SWL2020 (invA).
Bacteriophage KB1int was used to transduce the
invA::cat mutation into SR-11 3041
(wt), yielding strain AWM472 (invA).
Allelic exchange was performed to disrupt the serotype Typhimurium
sipB gene. A fragment of the sipB gene was
amplified from serotype Typhimurium SR-11 (wt) using primers
5'-GAAGGTACCGAAGATGAGTCTCTGCGG-3' and
5'-GAGCTCTTCTCAACAGAATGAT-3', which generated a PCR fragment with unique 5' KpnI and 3' SacI sites,
respectively. The resulting PCR product was blunt-end ligated into the
EcoRV site of pBluescript SK(+) and sequenced to verify its
accuracy. Subsequently, the sipB allele was inactivated by
insertion of a chloramphenicol resistance gene (a 1.2-kb
SmaI fragment from pCMXX [7]) into a unique
SmaI site. This plasmid was restricted with KpnI
and SacI, and the insertionally mutagenized
sipB::cat allele was cloned into
suicide plasmid pJP5603 (42). The resulting plasmid was electroporated into E. coli S17pir
(31) and conjugated to AJB3, a nalidixic acid-resistant
derivative of serotype Typhimurium SR-11 (51). A
chloramphenicol- and nalidixic acid-resistant exconjugant was selected
and named SWL2025 (sipB). Bacteriophage KB1int
was used to transduce the sipB::cat
mutant allele into SR-11 3041 (wt) and AWM405 (ompR),
yielding strains AWM501 (sipB) and AWM499 (ompR
sipB), respectively.
Allelic exchange was performed to disrupt the serotype Typhimurium
ssrB gene. An 853-bp fragment of the ssrB allele
was amplified from serotype Typhimurium ATCC14028 (wt) using primers
5'-CTTAATTTTCGCGAGGGCAGC-3' and
5'-TAGAATACGACATGGTAAAGCCCG-3'. This PCR product was cloned into pCR-Blunt (Invitrogen, Carlsbad, Calif.). The ssrB
allele was inactivated upon insertion of a chloramphenicol resistance gene (a 1.2-kb SmaI fragment from pCMXX
[7]) into a unique SspI site. This plasmid
was digested with EcoRI, and the disrupted ssrB
allele was ligated into suicide vector pKAS32 (48). The resulting plasmid (pMJW99) was transformed into E. coli
SM10pir and conjugated to serotype Typhimurium ATCC 14028 derivative BA715 (rpsL) (1). A double crossover
at the ssrB allele was obtained via homologous
recombination. A chloramphenicol- and streptomycin-resistant exconjugant was selected and named MJW129 (ssrB).
Bacteriophage P22HTint was used to transduce the
ssrB::cat mutant allele into SR-11
3041 (wt) and AWM405 (ompR), yielding strains AWM527
(ssrB) and AWM543 (ompR ssrB), respectively.
Macrophage assays.
The murine derived macrophage cell lines
J774 (American Type Culture Collection [ATCC], Manassas, Va.) and
RAW264.7 (ATCC) were cultured (37°C, 5% CO2) in Dulbecco
modified Eagle medium (DMEM; Gibco-BRL, Rockville, Md.), supplemented
with 10% fetal bovine serum (FBS; Gibco-BRL), glutamine (Gibco-BRL),
sodium pyruvate (Gibco-BRL), and nonessential amino acids (Gibco-BRL).
Bone marrow-derived macrophages were isolated from C57BL/6 mice
(Jackson Laboratories, Bar Harbor, Maine) and cultured for 6 days
(37°C, 5% CO2) in DMEM supplemented with 10% FBS, 20%
L929 supernatant (a generous gift from H. G. A. Bouwer,
Immunology Research, VAMC, Portland, Oreg.), and glutamine and sodium
pyruvate (Gibco-BRL).
Macrophage survival assays (gentamicin protection assays) were
performed as described by Fields et al. (20). In brief,
105 J774 macrophages were infected with stationary-phase
cultures (below) at a multiplicity of infection (MOI) of 
1. At
18 h postinfection, monolayers were washed three times with
phosphate-buffered saline (PBS) and lysed with Triton X-100 (Sigma).
Bacterial viability was determined by plating for CFU at various times
postinfection. Similar results were obtained using RAW264.7 macrophages
(data not shown).
The percentage of macrophage cytotoxicity was determined by measuring
the release of host cytoplasmic lactate dehydrogenase (LDH). J774 and
RAW264.7 macrophages were infected with bacterial cultures grown to
either late-log phase or stationary phase (below) at an input MOI of
~60. At 1 h postinfection, infected monolayers were washed three
times with PBS and lysed with Triton X-100 (Sigma), after which
bacterial uptake was determined by plating for viable intracellular
CFU. Differences between strains were observed and taken into account
by normalizing to the number of internalized bacteria (approximately
1% of input bacteria). At 6 and 18 h postinfection, the release
of LDH was quantified colorimetrically using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.). The
absorbance (A490) was determined on a microplate
reader (Dynatech Laboratories, Inc., Chantilly, Va.), after which the
percentage of cytotoxicity was calculated using the following formula:
100 × [(experimental release 
spontaneous
release)/(maximum release spontaneous release)]. The
spontaneous release is the amount of LDH released from the cytoplasm of
uninfected macrophages, whereas the maximum release is the amount of
LDH present in whole-cell lysates from uninfected macrophages.
In addition to measuring the release of LDH, quantitative macrophage
cytotoxicity assays were performed as described by Lindgren et al.
(35; data not shown). In brief, to determine the MOI at which 50% of the infected macrophages are killed
(MOICD50), 105 J774 macrophages were infected
with twofold serial dilutions of bacterial cultures (31 MOI 1,000, the limits of detection), as verified by plating for
CFU. At 6 and 18 h postinfection, the remaining viable macrophages
were fixed in a 10% formalin solution (10 to 15 min) and stained in a
0.13% crystal violet solution (>2 h). The absorbance
(A595) was determined on a microplate reader (Dynatech Laboratories); the MOI for the well that gave 50% of the
absorbance recorded for uninfected wells was considered the MOICD50 (i.e., 50% of the cytotoxic dose). Similar results
were obtained using RAW264.7 macrophages (data not shown).
The Cell Death Detection ELISAPLUS Assay (Roche Diagnostics
Corp.) was used to determine whether serotype Typhimurium-infected macrophages were undergoing apoptosis. This assay has been used successfully to study Pseudomonas aeruginosa-induced
apoptosis in eukaryotic cells (26). Macrophages were
infected with bacterial cultures grown to either late-log phase (data
not shown) or stationary phase (below) at an infection rate of 1.5 bacteria per macrophage. The amount of cytoplasmically located histones
bound to fragmented DNA was quantified colorimetrically at 18 h
postinfection, after which the absorbance
(A410nm) was determined on a microtiter plate reader. An enrichment factor indicative of apoptosis was calculated using the following formula:
(A410[experimental])/(A410[uninfected]).
Bacterial cultures were grown under various conditions. To obtain
stationary-phase cultures, bacteria were grown aerobically in LB broth
(3 ml) for 15 h at 37°C. To obtain late-log phase cultures,
bacteria were grown overnight (aerobically, 15 h at 37°C) in LB
broth (3 ml), subcultured 1:20 in LB broth (3 ml), and grown to
late-log phase (3 h) under the same culture conditions. Using a
MudJ transcriptional fusion to sipB, optimal
transcription of SPI1 genes in late-log phase cultures was confirmed
since under these culture conditions high levels of 
-galactosidase
were produced (data not shown).
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RESULTS |
Serovar Typhimurium kills macrophages independently of SPI1.
Conflicting reports on macrophage killing (13, 35, 39)
prompted us to investigate the effect of bacterial growth phase on the
ability of serotype Typhimurium to kill macrophages. Throughout this
study, two complementary methods were used to determine
Salmonella-induced cell death in both J774 and RAW264.7
macrophages. In addition to measuring the release of cytoplasmic LDH,
macrophage killing was calculated using a quantified macrophage
cytotoxicity assay (data not shown) (35). Strikingly similar
results were obtained with these two independent assays.
Salmonella-induced macrophage cell death was determined by
measuring the release of LDH at infection rates of about 0.7 and 1.5 bacteria per macrophage. Other MOIs were also tested, with identical
results (data not shown).
Under SPI1-inducing conditions (see Materials and Methods)
(13), rapid, SPI1-dependent macrophage killing was
observed (Fig. 1A). In contrast,
bacterial cultures grown to stationary phase, while unable to rapidly
kill infected macrophages, induced a delayed cytotoxic effect (Fig.
1B). Delayed induction of macrophage cell death required neither
invA nor sipB (Fig. 1B) and was observed as early
as 12 to 13 h postinfection (Fig. 1C). These results suggest that
serotype Typhimurium induces delayed macrophage cell death
independently of SPI1.
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FIG. 1.
Serovar Typhimurium kills macrophages independently of
SPI1. J774 macrophages were infected with late-log-phase (A) or
stationary-phase (B) cultures of wt serotype Typhimurium or strains
carrying null mutations in invA or sipB.
Bacterial growth was monitored by measuring optical density at 600 nm
(see Materials and Methods; also, data not shown). (A and B) Macrophage
cell death was quantitated at 6 h (A) and 18 h (B)
postinfection by measuring the release of LDH. (C) Using
stationary-phase cultures of either wild-type serotype Typhimurium or
an invA-deficient strain, macrophage cytotoxicity was
monitored for 20 h and quantitated at 2-h intervals by measuring
the release of LDH. Data from the graphs in panels A and B are
arithmetic means of at least three independent experiments. Error bars
indicate the standard deviations of the mean. The data from graph C are
representative of two independent experiments.
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SPI2 and ompR are required for delayed macrophage
killing.
Delayed cytotoxicity was dependent on a functional
ompR locus, since ompR mutant bacteria were
unable to kill infected macrophages (Fig.
2A). Recent evidence suggests that OmpR
activates transcription of the SPI2 encoded regulon ssrAB
(32). This operon is essential for the transcription of SPI2
genes (14), which are highly induced inside macrophages
(16, 50). To test whether, in addition to ompR,
SPI2 is required for delayed induction of macrophage cell death,
serotype Typhimurium strains mutated in ssrB and
spiB were tested. These genes encode a transcriptional
activator and a structural component of the SPI2 encoded type III
protein export apparatus, respectively (41). As shown in
Fig. 2B, serotype Typhimurium strains mutated in ompR,
ssrB, or spiB were unable to kill infected
macrophages when grown to stationary phase prior to infection. However,
these strains were fully cytotoxic under SPI1 inducing conditions (Fig.
2C), indicating that ompR and SPI2 are not required for
rapid induction of macrophage cell death. Cumulatively, these results
suggest that delayed, SPI1-independent cytotoxic effects are masked
under conditions that turn on SPI1 gene expression.
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FIG. 2.
SPI2 and ompR are required for delayed
macrophage killing. (A) J774 macrophages were infected with
stationary-phase cultures of either wt serotype Typhimurium or an
ompR-deficient strain, after which macrophage cytotoxicity
was monitored for 20 h and quantitated at 2-h intervals by
measuring the release of LDH. (B and C) In addition, J774 macrophages
were infected with stationary-phase (B) or late-log-phase (C) cultures
of wild-type serotype Typhimurium or strains carrying null mutations in
either ompR, ssrB, or spiB. Macrophage
cell death was quantitated at 18 h (B) and 6 h (C)
postinfection by measuring the release of LDH. Data from the graph in
panel A are representative of two independent experiments. The data
from the graphs in panels B and C are arithmetic means of at least
three independent experiments. The error bars indicate the standard
deviations of the mean.
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In agreement with the literature, we observed a defect (2- to 10-fold)
in intracellular proliferation for SPI2 mutant strains at 15 h
postinfection (14, 27, 28, 41, 46). However, long-term
intracellular survival and proliferation is not required for delayed
macrophage killing per se, since a prc mutant, encoding a
periplasmic protease (6, 20) required for intracellular survival and growth (Fig. 3A) (11,
21), kills infected macrophages as efficiently as the wild type
(Fig. 3B). Thus, despite a profound macrophage survival defect, the
prc mutant was fully cytotoxic. In fact, the prc
mutant strain was representative of a large panel of serotype
Typhimurium mutants that are defective in intramacrophage survival and
yet were still cytotoxic (data not shown). Collectively, these
observations suggest that long-term intramacrophage survival and growth
are not required for delayed, ompR- and SPI2-dependent macrophage killing. However, an indirect effect can not be ruled out
until we have identified the SPI2 secreted effector(s) involved.
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FIG. 3.
Long-term intracellular survival and growth is not
required for delayed macrophage killing. (A) J774 macrophages were
infected with stationary-phase cultures (conditions shown to turn off
SPI1-dependent rapid induction of macrophage cell death) of either
wild-type serotype Typhimurium, a spiB mutant strain, or a
macrophage-sensitive prc-deficient strain, after which
macrophage survival was determined at 15 and 18 h postinfection
(three times each) by measuring the viable intracellular CFU. (B)
Macrophage cytotoxicity was quantitated at these times by measuring the
release of cytoplasmic LDH. The data are arithmetic means of at least
three independent experiments from 15-h time points. The error bars
indicate the standard deviations of the mean.
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Rapid and delayed macrophage killing processes are
independent.
To determine whether rapid and delayed macrophage
killing were independent of one another, doubly deficient mutant
strains were constructed. Double mutants carried null mutations in
genes required for either rapid macrophage killing only (invA
sipB), delayed macrophage killing only (ompR ssrB), or
genes required for both rapid and delayed macrophage killing
(ompR sipB and ssrB invA). Under SPI1 inducing
conditions, ompR sipB, invA sipB, and ssrB
invA doubly deficient mutants were noncytotoxic, whereas an
ompR ssrB double mutant was as cytotoxic as the wt (Fig.
4A). Under conditions that favored
delayed macrophage killing, an invA sipB doubly deficient
strain was fully cytotoxic, whereas ompR sipB, ompR
ssrB, and ssrB invA double mutants were unable to kill infected macrophages (Fig. 4B). To demonstrate that these observations were not specific to J774 macrophages, these results were confirmed using RAW264.7 macrophages (data not shown) and bone marrow-derived macrophages (Fig. 5).
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FIG. 4.
Rapid and delayed macrophage killing processes are
independent. J774 macrophages were infected with wt serotype
Typhimurium or ompR sipB, ompR ssrB, invA
sipB, or ssrB invA double mutants. (A and B) Bacterial
cultures were grown to either late-log phase (A) or stationary phase
(B) prior to infection. Macrophage cell death was quantitated at 6 h (A) and 18 h (B) postinfection by measuring the release of LDH.
The data from each graph are arithmetic means of at least three
independent experiments. The error bars indicate the standard
deviations of the mean.
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FIG. 5.
S. typhimurium induces rapid and delayed
macrophage cell death in bone marrow-derived macrophages. To
demonstrate that serotype Typhimurium induced rapid and that delayed
macrophage cell death was not specific to J774 macrophages, these
results were repeated in RAW264.7 macrophages (data not shown). In
addition, bone marrow-derived macrophages were established from C57BL/6
mice and infected with mutant strains defective in inducing either
rapid macrophage cell death (sipB, invA sipB) or
delayed macrophage cell death (ompR, ompR ssrB)
or with a mutant strain defective in both rapid and delayed macrophage
killing (ssrB invA). (A and B) Bacterial strains were grown
to either late-log phase (A) or stationary phase (B) prior to
infection. Macrophage cell death was quantitated at 6 h (A) and
30 h (B) postinfection by measuring the release of LDH. The data
from each graph are arithmetic means of three independent experiments.
The error bars indicate the standard deviations of the mean.
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Collectively, these results indicate that bacterial strains mutated in
genes required for either rapid or delayed induction of macrophage cell
death are noncytotoxic only under specific pregrowth conditions.
However, bacterial strains mutated in loci that affect both rapid and
delayed macrophage killing are noncytotoxic under all conditions
tested. These observations are evidence that rapid and delayed
macrophage killing processes act independently of one another.
ompR and SPI2, but not SPI1, are required for delayed
induction of apoptosis in infected macrophages.
Next, we
investigated the nature of serotype Typhimurium-induced rapid and
delayed macrophage cell death. Thus far, a nonspecific method,
measuring the release of host cytoplasmic LDH, was used to calculate
macrophage cytotoxicity. To determine whether macrophages were
undergoing apoptosis upon infection with serotype Typhimurium, the
amount of cytoplasmically located histones bound to fragmented DNA was
quantified. Under SPI1 inducing conditions, serotype Typhimurium rapidly induced apoptosis via an SPI1-dependent process (data not
shown). Under conditions that favored delayed macrophage cytotoxicity, killing was independent of SPI1 (Fig. 6).
Delayed induction of apoptosis was abrogated in strains defective in
either ompR or SPI2 (Fig. 6). These results indicate that
serotype Typhimurium induces either rapid or delayed apoptosis in
infected macrophages. Rapid activation of programmed cell death depends
on SPI1, whereas delayed induction of apoptosis is SPI1 independent.
Furthermore, our observations suggest that ompR and SPI2 are
required for delayed activation of programmed macrophage cell death.
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FIG. 6.
ompR and SPI2, but not SPI1, are required for
delayed induction of apoptosis in infected macrophages. J774
macrophages were infected with wt serotype Typhimurium or with mutant
strains defective in either rapid killing (sipB,
invA, invA sipB) or delayed killing
(ompR, ssrB, spiB, ompR
ssrB) with a mutant strain defective in both rapid and delayed
macrophage killing (ssrB invA) or with a strain defective in
macrophage survival (prc). The ability of these strains to
induce apoptosis was determined at 18 h postinfection by measuring
the amount of cytoplasmically located histones bound to fragmented DNA.
The data from this graph are the arithmetic means of three independent
experiments. The error bars indicate the standard deviations of the
mean.
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DISCUSSION |
In this study, we demonstrate that macrophages undergo either
rapid or delayed apoptosis upon infection with serotype Typhimurium. Delayed activation of programmed cell death is masked when SPI1 genes
are expressed. Mutations that affect either rapid or delayed induction
of apoptosis result in noncytotoxic phenotypes only under specific
growth conditions. However, mutants defective in both rapid and delayed
macrophage killing are unable to induce apoptosis under any condition
tested, even at a high MOI (data not shown). Rapid activation of
programmed macrophage cell death depends on SipB and the SPI1 encoded
type III protein export machinery, whereas delayed induction of
apoptosis is SPI1 independent. Our results indicate that
ompR and a functional SPI2 encoded type III protein
secretion system are required for delayed induction of apoptosis.
However, a nonspecific effect cannot be excluded until we have
identified an SPI2 effector(s) that is both necessary and sufficient
for the activation of delayed programmed macrophage cell death.
In agreement with the literature, we observed a defect (2- to 10-fold)
in intracellular proliferation for SPI2 mutants at 15 and 18 h
postinfection (14, 27, 28, 41, 46). However, prc,
htrA, and 11 other macrophage-sensitive mutants tested are fully cytotoxic and yet are more severely defective in their ability to
survive and grow inside phagocytic cells (Fig. 3A) (11, 20). In fact, MS4290 (prc) was the most sensitive mutant isolated
in an extensive search for Salmonella mutants that cannot
survive inside macrophages (11, 20). Despite this
substantial defect, prc mutant bacteria, as well as a large
panel of other macrophage-sensitive serotype Typhimurium mutants,
induced both rapid (data not shown) and delayed apoptosis in infected
macrophages (Fig. 3B and Fig. 6). These results strongly support an
additional role for SPI2 in delayed induction of apoptosis in infected macrophages.
Our observations indicate that rapid and delayed activation of
programmed macrophage cell death are independent of one another, since
mutations in SPI1 do not affect delayed induction of apoptosis and
mutations in SPI2 do not affect rapid induction of apoptosis. Recent
studies support this view by demonstrating that these two specialized
protein secretion systems are controlled by distinct regulatory
circuits. For example, substrates for the SPI1 encoded type III protein
export apparatus are secreted under mildly alkaline conditions
(15), whereas substrates for the type III protein export
system encoded within SPI2 are secreted at pH 5.0 (9). Furthermore, numerous studies suggest that, once inside a phagocytic host, serotype Typhimurium represses SPI1 gene expression and turns on
genes that are important for long-term residence, growth, and survival
inside these host cells (2, 5, 8, 14, 16, 19, 24, 25, 33, 34, 43,
44, 50). It is therefore unlikely that substrates for SPI1 and
SPI2 encoded type III protein export systems are secreted simultaneously.
Our hypothesis is that serotype Typhimurium induces rapid and delayed
apoptosis in infected macrophages under discrete physiological conditions at distinct times and locations during the natural course of
infection in the host (Fig. 7).
Accumulating evidence suggests that the SPI1 encoded type III protein
secretion system is important primarily during the intestinal phase of
infection, since SPI1 mutants are significantly attenuated only when
administered to mice orally (reference 22 and
references therein and reference 23). In contrast,
ompR and SPI2 are absolutely required during the systemic
phase of infection (12, 16, 17, 41, 47, 50). In fact, SPI2
has been implicated in growth inside phagocytic cells at systemic sites
of infection (12, 16, 17, 41, 47, 50). A possible
consequence of the rapid, SPI1-dependent induction of apoptosis in
macrophages of the GALT is that additional phagocytic cells are
attracted to the site of inflammation. Our model suggests that
Salmonella represses the SPI1-dependent killing mechanism
upon internalization by macrophages, allowing continued proliferation
and systemic spread prior to ompR- and SPI2-dependent induction of delayed apoptosis at systemic sites of infection. Because
apoptotic cells are ingested by neighboring phagocytes, we propose that
delayed induction of apoptosis in infected macrophages may allow
Salmonella to spread intercellularly within apoptotic bodies. This model is supported by a recent study in which it was
demonstrated that serotype Typhimurium is transported from the
intestine, via the bloodstream, to the liver and spleen by CD18-expressing monocytes in an SPI1-independent process
(52), as well as by studies in which it was demonstrated
that Salmonella virulence was unaffected by treatment with
antibiotics that kill extracellular bacteria (10, 18).
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FIG. 7.
Model of serotype Typhimurium-induced apoptosis in vivo.
We propose that serotype Typhimurium induces rapid and delayed
apoptosis in infected macrophages under discrete physiological
conditions at distinct times and locations during the natural course of
infection. Because the SPI1 encoded type III protein secretion system
is important primarily during the intestinal phase of infection
(23), we propose that rapid, SPI1-dependent induction of
apoptosis in macrophages of the GALT results in increased inflammation
and recruitment of phagocytes that may be required for systemic
dissemination. Our model predicts that Salmonella represses
the rapid macrophage killing mechanism upon internalization, permitting
extensive intracellular proliferation and systemic spread prior to
delayed, ompR- and SPI2-dependent induction of apoptosis at
systemic sites of infection. In support of this view, ompR
and SPI2, unlike SPI1, are required during the systemic phase of
infection (12, 16, 17, 41, 47, 50). This model predicts that
Salmonella induces delayed apoptosis in infected macrophages
to spread intercellularly within apoptotic bodies.
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We thank Renée Tsolis for providing strain STN119 prior to
publication and Joanne Engel for helpful discussions. L929 supernatant was generously provided by Archie Bouwer. We thank members of the
Heffron and So laboratories for critical comments on the manuscript.
This work was supported by Public Health Service grant AI37201 to F.H.
from the National Institutes of Health.
| 1.
|
Ahmer, B. M.,
J. van Reeuwijk,
P. R. Watson,
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Abstract
Introduction
